Unique Mechanistic Insights Into The Beneficial Effects Of Angiotensin-(1-7) On The Prevention Of Cardiac Fibrosis: A Metabolomic Analysis Of Primary Cardiac Fibroblasts

Background: Cardiac fibrosis is a significant global health problem associated with almost all types of cardiovascular disease.Cardiac fibroblasts are the major effector cells of cardiac fibrosis.Cell metabolic pathways are highly conserved among species and change rapidly in response to drug stimulation.Currently,the examination of drug-induced metabolic pathways in disease cell model has been recommended by the Food and Drug Adinistration to speed the drug development process.Hence,we try to dissect the effects of angiotensin-(1-7)in a primary cell model of cardiac fibrosis established in angiotensin II-stimulated cardiac fibroblasts via metabolomics,and further clarify the potential protective mechanism of angiotensin-(1-7).Methods and Results: After exposing cardiac fibroblasts to angiotensin II and/or angiotensin-(1-7),172 metabolites in these cells and 129 in their culture medium were quantified and identified by gas chromatography-mass spectrometry.For the intracellular metabolites,the data were analyzed by orthogonal partial least squares discriminant analysis to shortlist biochemically significant metabolites associated with the antifibrotic action of angiotensin-(1-7).Seven significant metabolites were identified: 10,13-dimethyltetradecanoic acid,arachidonic acid(AA),aspartic acid,docosahexaenoic acid,glutathione,palmitelaidic acid,and pyroglutamic acid.By metabolic network analysis,we found that these metabolites were involved in six metabolic pathways,including arachidonic acid metabolism,leukotriene metabolism,and the γ-glutamyl cycle.Unlike the analysis of intracellular metabolites,the extracellular were analyzed by weighted coexpression network analysis(WGCNA).The WGCNA analysis yield the detection of 2 modules highly related to the protective action of angiotensin-(1-7)(r = 0.5 and 0.52;p < 0.05).By network-based enrichment analysis,we revealed that the 2 metabolite modules were enriched for 23 KEGG pathways such as renin-angiotensin system,cysteine and methionine metabolism,glycine,serine and threonine metabolism,valine,leucine and isoleucine biosynthesis,biosynthesis of unsaturated fatty acids,alanine,and aspartate and glutamate metabolism.Since the above intreseting pathways are related to calcium balance and oxidative stress,we further performed traditional molecular biology experiments in vivo and in vitro and verified that angiotensin-(1-7)inhibited the AA-TRPV4 mediated intracellular calcium overload and excessive accumulation of intracellular reactive oxygen species(ROS)origining from Nox4 in angiotensin II-stimulated cardiac fibroblasts.More importantly,we found that angiotensin-(1-7)suppressed the abnormal calcium-and ROSdependent activation of CaMKIIδ,the increased expression of CaMKIIδ-related proteins(CTGF,and ERK1/2),unlimited cardiac fibroblast activation,and excessive collagen deposition.Conclusions: Angiotensin-(1-7)can ameliorate the angiotensin IIstimulated metabolic perturbations associated with cardiac fibroblast activation.These metabolic changes indicate that the modulation in the calcium-and ROS-dependent activation of CaMKIIδ mediates the activity of angiotensin-(1-7)against cardiac fibrosis.Moreover,glutathione,pyroglutamic acid and arachidonic acid could be potential biomarkers for monitoring the antifibrotic action of angiotensin-(1-7).